A new shield for a cytokine storm

Summary

Highly virulent influenza virus infection results in excessive cytokine production, recruitment of leukocytes and immune-mediated pulmonary injury. Teijaro et al., 2011 now demonstrate that sphingosine 1 phosphate receptor 1 ligands suppress all features of flu-inflicted pathological inflammation and places the endothelium at the center of this regulatory network.

Recognition and rapid clearance of pathogens by the innate immune system provides the first line of defense in metazoan organisms. However, excessive activation of the innate immune system in response to pathogens can lead to pathological inflammatory consequences. In the case of highly virulent 1918 and avian H5N1 influenza virus infections, early recruitment of inflammatory leukocytes to the lung, followed by excessive early cytokine responses (known as a cytokine storm), is considered to be the key contributor to morbidity and mortality of the infection (Tscherne and Garcia-Sastre, 2011). Likewise, the virulence of the pandemic 2009 H1N1 “swine flu” is associated with viral replication in the lower respiratory tract and more severe pulmonary damage compared to seasonal flu (Tscherne and Garcia-Sastre, 2011). However, the cell types responsible for the initiation and amplification of the cytokine storm that follow virulent influenza infection remain unclear. In this issue, Teijaro et al. have uncovered an unexpected role of endothelial cells in coordinating the inflammatory sequelae via sphingosine-1 phosphate signalling.

Sphingosine-1 phosphate is a metabolite of sphingolipid and is a ligand for a family of five G-protein coupled receptors, S1P_1-5 (Rosen et al., 2009). Differential expression of these receptors enables control of angiogenesis, heart development, and immunity in a highly specific manner. The sphingosine analogue FTY720, is a well-known immunomodulator that has recently been approved for the treatment of multiple sclerosis (Brinkmann et al., 2010). FTY720 is phosphorylated in vivo by sphingosine kinase 2 to produce a ligand for S1P receptors, S1P_{1, 3-5}. Its mode of action is through sequestration of lymphocytes in lymph nodes away from peripheral sites of inflammation. Prolonged exposure to FTY720 causes S1P₁ endocytosis and degradation, impairing the ability of lymphocytes to respond to endogenous S1P to egress out of lymph nodes (Cyster, 2005).

The study by Teijaro et al., 2011) followed up on the previous findings by the same group that local intratracheal instillation of S1P ligands reduces cytokine responses following respiratory infections with a mouse adapted influenza virus (Marsolais et al., 2008; Marsolais et al., 2009) or human 2009 H1N1 influenza isolate (Walsh et al., 2011). These studies also revealed that the S1P₁-specific agonists do not affect the generation of adaptive immune responses (CD8 T cells and neutralizing antibodies) and do not alter viral replication in vivo (Marsolais et al., 2008). Using S1P₁-specific agonists, the current study shows that stimulation of S1P₁ alone recapitulates much of the suppressive phenotype of AAL-R, which is a broad-spectrum agonist of S1P receptors. Notably, intratracheal instillation of CYM-5442 (an S1P₁-agonist) on hours 1, 13, 25 and 37 of influenza virus challenge significantly dampened type I IFN, cytokine and chemokine release into the bronchoalveolar lavage (BAL), and resulted in a significant decrease in infiltration of monocytes, macrophages, neutrophils and natural killer (NK) cells into the lung. In addition, oral administration of another S1P₁ agonist, RP-002, at 1 and 25 hours after infection resulted in increased survival of mice challenged with 2009 H1N1 human isolate (Figure 1). To address the mechanism of action, the authors examined the expression of S1P₁ using S1P₁-eGFP knock-in mice (Cahalan et al., 2011). As reported previously, in addition to lymphocytes, high level of S1P₁ expression was found on lymphatic and vascular endothelial cells isolated from the lung (Cahalan et al., 2011). However, influenza-induced cytokine responses and cellular recruitment to the lung were blocked by CYM-5442 in RAG2-deficient mice (devoid of T and B lymphocytes), suggesting that S1P1 stimulation dampens innate immune responses in a manner independent of its well-known function in lymphocyte sequestration.

Figure. Suppression of inflammation by S1P1 agonists.

Influenza virus infects the lung epithelial cells and subsequently alveolar macrophages, and other leukocytes. This results in type I IFN, cytokine and chemokine secretion by various cell types both resident and recruited. S1P₁ ligands, CYM05442 and RP-002, engage S1P₁ on vascular and lymphatic (not shown) endothelial cells and block chemokine secretion from endothelial cells, which in turn block recruitment of leukocytes to the lung. In addition, S1P₁ signaling suppresses type I IFN, cytokine and chemokine secretion from other cells, resulting in reduced immunopathology. However, S1P1 agonists do not impair dendritic cell activation, thereby enabling T cell and B cell responses to be initiated in the draining lymph node.

Next, the authors demonstrated that production of chemokines, CCL2, CCL5 and CXCL10, from vascular and lymphatic endothelium following influenza infection was significantly reduced by CYM-5442 treatment in vivo. Intratracheal instillation of recombinant CCL2 restored monocyte, macrophage and NK cell recruitment to the lung of mice treated with CYM-5442. Surprisingly, CCL2 was not sufficient to restore IFN or cytokine responses in the lung. Further, depletion of CD11b+ cells (including monocytes, macrophages, neutrophils and NK cells) using antibody treatment only resulted in reduction in IFN-γ secretion but did not affect the levels of IFN-α, CCL2, IL-6 or TNF-α in the lung. These data indicate that inflammatory leukocyte recruitment is not sufficient for cytokine storm in the face of CYM-5442 treatment, and is not required for the production of majority of the cytokines in response to influenza infection. Finally, type I IFNs were placed upstream of the cytokine storm during influenza infection, as IFNαβR-deficient mice failed to secrete type I IFN, chemokines or cytokines despite the normal recruitment of inflammatory leukocytes.

The results of this study reveal an important role of S1P₁ as a regulator of inflammation but also raise a host of questions. First, how does S1P₁ agonism result in global suppression of cytokines? This may occur both at the cell intrinsic level within the lung endothelial cells and at the cell extrinsic level in the recruited leukocytes. S1P₁ receptor is coupled to Gαi and receptor engagement triggers a multitude of downstream signaling pathways, including PI3K and Rac activation and promoting cell survival, motility and barrier functions. S1P₁ also activates the MAP kinase and phospholipase C pathways and intracellular mobilization of calcium signaling resulting in cell proliferation and cytokine secretion (Rosen et al., 2009). However, it remains unclear whether any of these pathways directly inhibit cytokine and chemokine production by endothelial cells, or alternatively, whether S1P₁-activated endothelial cells produce anti-inflammatory mediators. It is also possible that the anti-inflammatory effect of S1P₁ engagement is an indirect consequence of its effect on vascular integrity.

Second, why doesn't the endogenous S1P in the blood or lymph, which is maintained at high concentration (100–1,000 nM in blood and 30–300 nM in lymph), trigger a similar response in the endothelial cells? Unlike S1P, the synthetic agonists of S1P₁, including CYM-5442, induce prolonged signaling, polyubiquitination of S1P₁ followed by degradation in the lysosomes (Rosen et al., 2009). Whether endocytosis and degradation of S1P₁ is requisite for immunosuppression by CYM-5442 and other S1P₁ agonists remains to be determined. In this regard, it is interesting to speculate whether similar blockade of inflammation is caused by FTY720, and whether the clinical effects of this compound may in part depend on mechanisms that extend beyond lymphocyte sequestration.

Following seasonal influenza infection, the virus replicates primarily in the lung epithelium, followed by infection of alveolar macrophages, dendritic cells, NK cells and B cells (Manicassamy et al., 2010) (Figure 1). In contrast, H5N1 avian flu targets specifically endothelial cells for replication in birds (Feldmann et al., 2000). Thus, it would be interesting to examine whether S1P₁ agonists will be effective in the case of H5N1 influenza. Finally, the possible role of endothelial S1P₁ in other conditions associated with excessive cytokine production would be an exciting question for future studies.